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Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

Rydman EM, Ilves M, Koivisto AJ, Kinaret PA, Fortino V, Savinko TS, Lehto MT, Pulkkinen V, Vippola M, Hämeri KJ, Matikainen S, Wolff H, Savolainen KM, Greco D, Alenius H - Part Fibre Toxicol (2014)

Bottom Line: However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

View Article: PubMed Central - PubMed

Affiliation: Nanosafety Research Centre, Finnish Institute of Occupational Health, Helsinki, Finland. elina.rydman@ttl.fi.

ABSTRACT

Background: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.

Methods: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation.

Results: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.

Conclusions: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

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Related in: MedlinePlus

Inhalation to rCNT induces AHR, mucus secretion and induction of Th2 type cytokines in the lungs. a: BALB/c mice exposed to rCNT for 4 h/day on 4 consecutive days showed enhanced AHR to metacholine on day 5. b-h: using the same exposure schedule, C57BL/6 mice were exposed to tCNT or rCNT and sacrificed on the following day. b: PAS-stained lung sections were used to determine the numbers of mucin-producing goblet cells. c-e: representative images of PAS-stained lung tissue of an untreated mouse (c) and mice exposed to tCNT (d) and rCNT (e) that show markable activation of mucin-producing cells in lungs of rCNT-exposed but not in tCNT-exposed mice. f, g: the mRNA expression levels measured in lung tissue revealed an upregulation of Th2 type cytokines Il-13 and Il-5 (f) and downregulation of Th1 cytokine Ifn-γ (g) after exposure to rCNT. h: furthermore, mRNA expression of eosinophil-attracting chemokines Ccl11, Ccl24 and Ccl17 was triggered in lungs by rCNT. Results of AHR measurements (a) are expressed as mean enhanced pause values (Penh) ± SEM (n = 9-11). PAS + cell counts (b) represent the average of positive cells per 200 μm of bronchus surface counted from three bronchi per mouse in control group and six bronchi per mouse in CNT-exposed groups (n = 10-20). Images c-e are shown at x400 magnification with a 50 μm scale bar. mRNA expression levels measured by qRT-PCR are presented as fold changes compared to untreated control mice (n = 10-20). *P < 0.05; **P < 0.01; ***P < 0.001. MCh, metacoline; C, control group.
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Fig2: Inhalation to rCNT induces AHR, mucus secretion and induction of Th2 type cytokines in the lungs. a: BALB/c mice exposed to rCNT for 4 h/day on 4 consecutive days showed enhanced AHR to metacholine on day 5. b-h: using the same exposure schedule, C57BL/6 mice were exposed to tCNT or rCNT and sacrificed on the following day. b: PAS-stained lung sections were used to determine the numbers of mucin-producing goblet cells. c-e: representative images of PAS-stained lung tissue of an untreated mouse (c) and mice exposed to tCNT (d) and rCNT (e) that show markable activation of mucin-producing cells in lungs of rCNT-exposed but not in tCNT-exposed mice. f, g: the mRNA expression levels measured in lung tissue revealed an upregulation of Th2 type cytokines Il-13 and Il-5 (f) and downregulation of Th1 cytokine Ifn-γ (g) after exposure to rCNT. h: furthermore, mRNA expression of eosinophil-attracting chemokines Ccl11, Ccl24 and Ccl17 was triggered in lungs by rCNT. Results of AHR measurements (a) are expressed as mean enhanced pause values (Penh) ± SEM (n = 9-11). PAS + cell counts (b) represent the average of positive cells per 200 μm of bronchus surface counted from three bronchi per mouse in control group and six bronchi per mouse in CNT-exposed groups (n = 10-20). Images c-e are shown at x400 magnification with a 50 μm scale bar. mRNA expression levels measured by qRT-PCR are presented as fold changes compared to untreated control mice (n = 10-20). *P < 0.05; **P < 0.01; ***P < 0.001. MCh, metacoline; C, control group.

Mentions: Classical signs of asthma include mucus hypersecretion, AHR and expression of Th2 type cytokines. Since it is well known that AHR cannot be elicited in the C57BL/6 mouse strain [14], we used BALB/c mice to demonstrate that rCNT inhalation induces AHR to inhaled metacholine (Figure 2a; Cellular infiltration in BAL and cytokine/chemokine expression in the lung tissue of rCNT exposed BALB/c mice is given in Additional file 1). We also found that rCNT, but not tCNT, triggered significant activation of mucin-producing goblet cells 24 h after 4-day inhalation exposure in C57BL/6 mice (Figure 2b-e). In addition, up-regulation of Th2 type cytokines Il-13 and Il-5 transcriptional levels (Figure 2f) and down-regulation of Th1 cytokine Ifn-γ (Figure 2g) after exposure to rCNT were evidenced in lung tissue. Furthermore, mRNA expression of eosinophil-attracting chemokines Ccl11, Ccl24 and Ccl17 was triggered by rCNT (Figure 2h). tCNT treatment had no effect on the expression of Il-13, Ifn-γ and Ccl24, however, it suppressed Il-5 and elevated Ccl11 and Ccl17 production. These results show that inhaled rCNT cause AHR, mucus production and trigger production of Th2 type cytokines and eosinophil chemoattractants, and thereby suggest that rCNT fibers can elicit a condition similar to allergic airway inflammation.Figure 2


Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

Rydman EM, Ilves M, Koivisto AJ, Kinaret PA, Fortino V, Savinko TS, Lehto MT, Pulkkinen V, Vippola M, Hämeri KJ, Matikainen S, Wolff H, Savolainen KM, Greco D, Alenius H - Part Fibre Toxicol (2014)

Inhalation to rCNT induces AHR, mucus secretion and induction of Th2 type cytokines in the lungs. a: BALB/c mice exposed to rCNT for 4 h/day on 4 consecutive days showed enhanced AHR to metacholine on day 5. b-h: using the same exposure schedule, C57BL/6 mice were exposed to tCNT or rCNT and sacrificed on the following day. b: PAS-stained lung sections were used to determine the numbers of mucin-producing goblet cells. c-e: representative images of PAS-stained lung tissue of an untreated mouse (c) and mice exposed to tCNT (d) and rCNT (e) that show markable activation of mucin-producing cells in lungs of rCNT-exposed but not in tCNT-exposed mice. f, g: the mRNA expression levels measured in lung tissue revealed an upregulation of Th2 type cytokines Il-13 and Il-5 (f) and downregulation of Th1 cytokine Ifn-γ (g) after exposure to rCNT. h: furthermore, mRNA expression of eosinophil-attracting chemokines Ccl11, Ccl24 and Ccl17 was triggered in lungs by rCNT. Results of AHR measurements (a) are expressed as mean enhanced pause values (Penh) ± SEM (n = 9-11). PAS + cell counts (b) represent the average of positive cells per 200 μm of bronchus surface counted from three bronchi per mouse in control group and six bronchi per mouse in CNT-exposed groups (n = 10-20). Images c-e are shown at x400 magnification with a 50 μm scale bar. mRNA expression levels measured by qRT-PCR are presented as fold changes compared to untreated control mice (n = 10-20). *P < 0.05; **P < 0.01; ***P < 0.001. MCh, metacoline; C, control group.
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Related In: Results  -  Collection

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Fig2: Inhalation to rCNT induces AHR, mucus secretion and induction of Th2 type cytokines in the lungs. a: BALB/c mice exposed to rCNT for 4 h/day on 4 consecutive days showed enhanced AHR to metacholine on day 5. b-h: using the same exposure schedule, C57BL/6 mice were exposed to tCNT or rCNT and sacrificed on the following day. b: PAS-stained lung sections were used to determine the numbers of mucin-producing goblet cells. c-e: representative images of PAS-stained lung tissue of an untreated mouse (c) and mice exposed to tCNT (d) and rCNT (e) that show markable activation of mucin-producing cells in lungs of rCNT-exposed but not in tCNT-exposed mice. f, g: the mRNA expression levels measured in lung tissue revealed an upregulation of Th2 type cytokines Il-13 and Il-5 (f) and downregulation of Th1 cytokine Ifn-γ (g) after exposure to rCNT. h: furthermore, mRNA expression of eosinophil-attracting chemokines Ccl11, Ccl24 and Ccl17 was triggered in lungs by rCNT. Results of AHR measurements (a) are expressed as mean enhanced pause values (Penh) ± SEM (n = 9-11). PAS + cell counts (b) represent the average of positive cells per 200 μm of bronchus surface counted from three bronchi per mouse in control group and six bronchi per mouse in CNT-exposed groups (n = 10-20). Images c-e are shown at x400 magnification with a 50 μm scale bar. mRNA expression levels measured by qRT-PCR are presented as fold changes compared to untreated control mice (n = 10-20). *P < 0.05; **P < 0.01; ***P < 0.001. MCh, metacoline; C, control group.
Mentions: Classical signs of asthma include mucus hypersecretion, AHR and expression of Th2 type cytokines. Since it is well known that AHR cannot be elicited in the C57BL/6 mouse strain [14], we used BALB/c mice to demonstrate that rCNT inhalation induces AHR to inhaled metacholine (Figure 2a; Cellular infiltration in BAL and cytokine/chemokine expression in the lung tissue of rCNT exposed BALB/c mice is given in Additional file 1). We also found that rCNT, but not tCNT, triggered significant activation of mucin-producing goblet cells 24 h after 4-day inhalation exposure in C57BL/6 mice (Figure 2b-e). In addition, up-regulation of Th2 type cytokines Il-13 and Il-5 transcriptional levels (Figure 2f) and down-regulation of Th1 cytokine Ifn-γ (Figure 2g) after exposure to rCNT were evidenced in lung tissue. Furthermore, mRNA expression of eosinophil-attracting chemokines Ccl11, Ccl24 and Ccl17 was triggered by rCNT (Figure 2h). tCNT treatment had no effect on the expression of Il-13, Ifn-γ and Ccl24, however, it suppressed Il-5 and elevated Ccl11 and Ccl17 production. These results show that inhaled rCNT cause AHR, mucus production and trigger production of Th2 type cytokines and eosinophil chemoattractants, and thereby suggest that rCNT fibers can elicit a condition similar to allergic airway inflammation.Figure 2

Bottom Line: However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

View Article: PubMed Central - PubMed

Affiliation: Nanosafety Research Centre, Finnish Institute of Occupational Health, Helsinki, Finland. elina.rydman@ttl.fi.

ABSTRACT

Background: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.

Methods: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation.

Results: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.

Conclusions: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

Show MeSH
Related in: MedlinePlus